Bottom Line:
The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood.DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively.Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

ABSTRACTThe intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

fig7: Maturation of DP-GFP borders is cytochalasin D sensitive. (A) Wounded DP-GFP expressing A431 monolayers were imaged at 1-min intervals in the presence of cytochalasin D. Phase I accumulation of DP-GFP (arrows) at the cell–cell border and the formation of phase II particles (arrowheads) was observed in the presence of cytochalasin D. Phase III particle translocation was not observed. (B) Fluorescence border intensity of cytochalasin D treated cells (gray squares) over time was compared with DMSO treated cells imaged in parallel (black diamonds). An initial increase in pixel intensity was observed, but did not increase appreciably over time. A second increase in border fluorescence was not observed, supporting the role of particle translocation in border maturation. Bar, 10 μm.

Mentions:
Because DP that lacks an IF-binding domain can traffic to intercellular borders, frequently with more rapid kinetics, and IF–associated DP particles are closely associated with cortical actin (Green et al., 1987), we hypothesized that reorganization of the actin cytoskeleton might drive later phases of DP dynamics. To test this idea, we imaged DP-GFP–expressing cells over time after disrupting filamentous actin with cytochalasin D. Phase I occurred and phase II particles formed (Fig. 7 A and not depicted), but did not appear to translocate to borders (Fig. 7 A). The increase in fluorescence intensity normally associated with phase III was not observed (Fig. 7 B). These data suggest that IFs and microfilaments are both involved in regulating DP dynamics.

fig7: Maturation of DP-GFP borders is cytochalasin D sensitive. (A) Wounded DP-GFP expressing A431 monolayers were imaged at 1-min intervals in the presence of cytochalasin D. Phase I accumulation of DP-GFP (arrows) at the cell–cell border and the formation of phase II particles (arrowheads) was observed in the presence of cytochalasin D. Phase III particle translocation was not observed. (B) Fluorescence border intensity of cytochalasin D treated cells (gray squares) over time was compared with DMSO treated cells imaged in parallel (black diamonds). An initial increase in pixel intensity was observed, but did not increase appreciably over time. A second increase in border fluorescence was not observed, supporting the role of particle translocation in border maturation. Bar, 10 μm.

Mentions:
Because DP that lacks an IF-binding domain can traffic to intercellular borders, frequently with more rapid kinetics, and IF–associated DP particles are closely associated with cortical actin (Green et al., 1987), we hypothesized that reorganization of the actin cytoskeleton might drive later phases of DP dynamics. To test this idea, we imaged DP-GFP–expressing cells over time after disrupting filamentous actin with cytochalasin D. Phase I occurred and phase II particles formed (Fig. 7 A and not depicted), but did not appear to translocate to borders (Fig. 7 A). The increase in fluorescence intensity normally associated with phase III was not observed (Fig. 7 B). These data suggest that IFs and microfilaments are both involved in regulating DP dynamics.

Bottom Line:
The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood.DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively.Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

ABSTRACTThe intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.